Tau isoform-specific enhancement of L-type calcium current and augmentation of afterhyperpolarization in rat hippocampal neurons

Accumulation of tau is observed in dementia, with human tau displaying 6 isoforms grouped by whether they display either 3 or 4 C-terminal repeat domains (3R or 4R) and exhibit no (0N), one (1N) or two (2N) N terminal repeats. Overexpression of 4R0N-tau in rat hippocampal slices enhanced the L-type calcium (Ca2+) current-dependent components of the medium and slow afterhyperpolarizations (AHPs). Overexpression of both 4R0N-tau and 4R2N-tau augmented CaV1.2-mediated L-type currents when expressed in tsA-201 cells, an effect not observed with the third 4R isoform, 4R1N-tau. Current enhancement was only observed when the pore-forming subunit was co-expressed with CaVβ3 and not CaVβ2a subunits. Non-stationary noise analysis indicated that enhanced Ca2+ channel current arose from a larger number of functional channels. 4R0N-tau and CaVβ3 were found to be physically associated by co-immunoprecipitation. In contrast, the 4R1N-tau isoform that did not augment expressed macroscopic L-type Ca2+ current exhibited greatly reduced binding to CaVβ3. These data suggest that physical association between tau and the CaVβ3 subunit stabilises functional L-type channels in the membrane, increasing channel number and Ca2+ influx. Enhancing the Ca2+-dependent component of AHPs would produce cognitive impairment that underlie those seen in the early phases of tauopathies.

It has been common to study transgenic animals as models of tauopathies 20 . However, most transgenic models cannot faithfully mimic sporadic diseases. Sporadic Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles of hyperphosphorylated tau 21 . There are six isoforms of tau in the human CNS. Isoforms are named after the number of near-N-terminal inserts (0N, 1N, 2N) and C-terminal repeats (3R and 4R) 22 . The balance between 3 and 4R isoforms of tau can change, with the ratio of 4R to 3R isoforms increased in the hippocampus and midfrontal cortex of AD patients 23 . Increasing expression of 4R relative to 3R isoforms of tau caused severe seizures and nesting behaviour abnormalities in mice 24 , while overexpression of human 2N4R-tau produced synaptic dysfunction and memory deficits 25 . However, the effect of acute expression of 4R-tau isoforms on neuronal excitability has yet to be determined. Here, we report that overexpression of the most abundant isoform in aged human brain, 4R0N-tau 26 , augments AHPs following a train of action potentials. This effect was specific on both AHP components that are dependent on Ca 2+ entry through L-type channels. Expression of 4R0N-or 4R2N-tau, but not 4R1N-tau, augmented Ca V 1.2-mediated L-type channel current in a Ca V β-specific manner. Enhancement of Ca V 1.2-mediated current resulted from an increase in the number of functional channels, and only occurred when the L-type channel was comprised of Ca V β3 and not Ca V β2a subunits. Co-immunoprecipitation of 4R0N-tau and Ca V β3 suggests that the two proteins might be in direct contact. These data demonstrate that accumulation of 4R-tau isoforms in hippocampal neurons increases the number of functional L-type Ca 2+ channels in the membrane, increasing Ca 2+ influx. This increased influx of Ca 2+ enhances the Ca 2+ -dependent components of AHPs that would reduce action potential firing and lead to cognitive decline 25 .

Results
Expression of 4R0N-tau augments AHPs in hippocampal neurons. The effect of 4R0N-tau expression on the medium and slow AHPs was examined in CA1 pyramidal neurons in organotypic hippocampal slices 24 h after slices were transduced with control EGFP-alone or EGFP + 4R0N-tau. The amplitude of both AHPs and the membrane currents underlying them were compared between control cells that over-expressed EGFP alone, and 4R0N-tau cells that expressed both the tau isoform and the marker EGFP.
Finally, we wished to confirm that overexpression of human 4R0N-tau enhances the Ca 2+ -dependent SK channel-mediated component of the medium AHP. Our previous data demonstrated that the SK channel component of the medium AHP was comprised of homomeric SK2 channels, which were blocked by apamin with an IC 50 of approximately 55 pM 27 . The medium AHP was isolated by dialyzing neurons with an electrode solution supplemented with cAMP (1 mM) to block the slow AHP 27 . Application of apamin (100 nM) reduced the amplitude of the medium AHP by 28.0 ± 8.0% in control cells (− 4.4 ± 0.5 vs − 3.1 ± 0.4, n = 3. AHP evoked by 15 action potentials) (Fig. 2C,E). The addition of apamin (100 nM) to 4R0N-tau neurons produced a greater inhibition of the afterpotential (49.5 ± 17.2%; − 4.9 ± 1.0 vs − 2.1 ± 0.6, n = 5. AHP evoked by 15

The 4R0N-tau-evoked enhanced Ca 2+ -dependent component of AHPs is mediated by L-type
Ca 2+ channels. The Ca 2+ -activated potassium channels underlying the two components of AHP in CA1 hippocampal pyramidal neurons are known to be different, with activation of SK channels contributing to the medium AHP 27 and a channel of unknown identity underlying the slow AHP [but see 7]. It is unlikely that two different types of Ca 2+ activated channel would be affected directly by expression of tau; instead, it is more likely that augmentation of the Ca 2+ -dependent components of AHPs results from an increase in Ca 2+ entry. The Ca 2+ -dependent component of the medium AHP and the slow AHP is suppressed by the dihydropyridine nimodipine 8,10,11 . The observation that the Ca 2+ -dependent potassium currents underlying the two AHP components are insensitive to blockers of other Ca 2+ channel subtypes indicates that they are solely dependent on Ca 2+ entry through L-type channels 11 . Therefore, it is possible that overexpression of 4R0N-tau enhances L-type Ca 2+ current to produce augmentation of both the Ca 2+ -dependent component of the medium AHP and the entire slow AHP. Both I mAHP and I sAHP were sensitive to nimodipine in control cells (n = 5), with 10 μM nimodipine reducing I mAHP by 29.7 ± 6.1% (I mAHP 164. www.nature.com/scientificreports/ augmentation of both current components by 4R0N-tau resulted from nimodipine-sensitive current. These data suggest that expression of 4R0N-tau enhances Ca 2+ entry through L-type channels to increase the amplitude of the Ca 2+ -dependent currents that underlie the medium and slow AHPs. Augmentation of L-type channel current by 4R0N-tau. Hippocampal neurons express both Ca V 1.2 and 1.3 isoforms of L-type Ca 2+ channels 28 , which are pharmacologically indistinguishable under voltage-clamp conditions within a brain slice 29 . Therefore, we elected to determine the effect of different tau isoforms on expressed recombinant channel current. Expression of the pore-forming subunit Ca V 1.2 and auxiliary Ca V α2δ1 and Ca V β3 subunits in tSA-201 cells produced inward Ca 2+ -carried current that activated from − 30 mV, peaking around + 10 mV. Evoked current displayed prominent decay during the 250 ms depolarizing voltage step (Fig. 4). Co-expression of 4R0N-tau produced Ca V 1.2-mediated currents of larger amplitude (Fig. 4B). Normalising current amplitude to cell capacitance showed that co-expression of functional Ca V 1.2 channels with 4R0N-tau enhanced current throughout the voltage range (Fig. 4C). Cells expressing Ca V 1.2, Ca V α2δ1 and Ca V β3 subunits produced whole-cell Ca 2+ current that had a peak amplitude of − 28.0 ± 5.6 pA/pF at + 20 mV (n = 19). Co-expression of 4R0N-tau augmented peak Ca V 1.2-mediated current, giving a peak current at + 20 mV of − 59.4 ± 12.1 pA/pF (n = 19) (p = 0.2) (Fig. 4E). Construction of peak current activation curves showed that augmentation of current by 4R0N-tau was not accompanied by a change in voltage dependence of activation (Fig. 4D) Neuronal L-type calcium current is also mediated by channels containing the pore-forming subunit Ca V 1.3. Co-expression of the Ca V 1.3 subunit with auxiliary Ca V α2δ1 and Ca V β3 subunits produced a current that activated more negative (− 40 mV) and showed a greater degree of current decay during the depolarizing voltage step than seen with Ca V 1.2/Ca V α2δ1/Ca V β3-mediated current 29,30 (Fig. 4F). Currents peaked approximately 30 mV more negative (at − 10 mV) than that derived from expression of Ca V 1.2/Ca V α2δ1/Ca V β3 subunits (Fig. 4H). Peak amplitude at − 10 mV was − 43.5 ± 13.6 pA/pF (n = 8) (Fig. 4J). Co-expression of 4R0N-tau augmented Ca V 1.3/Ca V α2δ1/Ca V β3-mediated inward current (Fig. 4G,H,J), increasing current density to − 58.9 ± 21.2 pA/pF (n = 6) (Fig. 4J). Enhanced current was not accompanied by changes in voltage dependence of activation ( Fig. 4I) (control V 0.5 − 11.1 ± 1.6 mV, 4R0N-tau V 0.5 : − 22.3 ± 9.3 mV). It is clear that there was a trend to increase current amplitude upon co-expression of 4R0N-tau, but the effect did not reach significance (p = 0.53).

Tau-mediated augmentation of L-type channel current is dependent on CaVβ. The most
prevalent Ca V β subunits in hippocampal neurons are Ca V β2a and Ca V β3 31 . We tested the effects of 4R0N-tau expression on Ca V β2a-containing Ca V 1.2 or 1.3 L-type channel current. Expression of Ca V 1.2, Ca V α2δ1 and Ca V β2a subunits in tSA-201 produced inward Ca 2+ current from − 30 mV (holding potential − 70 mV) (Fig. 5A). Co-expression of 4R0N-tau had no effect on the amplitude of evoked current (control peak current density − 17.3 ± 4.9 pA/pF, n = 5; 4R0N-tau-expressing − 16.0 ± 3.0 pA/pF) (p = 0.82) (Fig. 5B-D). The same lack of effect of co-expression of 4R0N-tau was observed on macroscopic current mediated by Ca V 1.3 channels that contained the Ca V β2a subunit (Fig. S3). Inward current was evoked from cells transfected and expressing Ca V 1.3, Ca V α2δ1 and Ca V β2a subunits from about − 40 mV and peaked around − 10 mV when Ca 2+ was the charge carrier and slightly more negative when Ba 2+ was used in place of Ca 2+ . Co-expression of these channel subunits with 4R0Ntau (Fig. S3) had little effect on current amplitude. These data indicate that augmentation of either Ca V 1.2 or 1.3-mediated current by co-expression of 4R-tau is dependent on the identity of the auxiliary Ca V β subunit.
Tau-mediated augmentation of L-type channel current results from an increase in functional channels. Augmentation of macroscopic current can involve increases in one or a combination of single channel current (i), open probability (Po) or the number of functional channels (N). It has been proposed that hippocampal L-type channel current increases in aged animals by an increase in the number of functional channels 18,19 . We used a non-stationary noise approach to estimate which of these parameters might underlie the increase in Ca V 1.2-mediated L-type current by co-expression of 4R0N-tau. Cells expressing Ca V 1.2, Ca V α2δ1 and Ca V β3 subunits were voltage-clamped at − 70 mV and subjected to a repeated depolarizing voltage step to the peak of the current-voltage relationship (+ 10 mV) (see Methods). This approach revealed that the coexpression of 4R0N-tau increased the amplitude of macroscopic current by increasing the number of functional channels (Fig. 5E). Analysis showed that the number of channels per cell significantly increased from 268 ± 76 in control EGFP-transfected cells (n = 7) to 1037 ± 215 in 4R0N-tau co-transfected cells (n = 7) (p = 0.03). Normalizing each cell estimate of channel number to the cell capacitance allowed us to estimate that channel density increased from 0.9 ± 0.2 channels/μm 2 in control cells to 2.5 ± 0.6 channels/μm 2 in cells co-expressing 4R0Ntau (Fig. 5E). Estimation of the amplitude of single channel current from this analysis showed that it did not increase, being − 0.80 ± 0.11 pA (n = 6) for control Ca V 1.2/Ca V α2δ1/Ca V β3-mediated current and − 0.58 ± 0.20 pA (n = 7) in cells co-expressing 4R0N-tau (Fig. 5F) (p = 0.39). Finally, co-expression of 4R0N-tau had no significant effect (p = 0.91) on channel open probability (Po) of Ca V 1.2/Ca V α2δ1/Ca V β3 channels (Fig. 5G). Cells transfected with Ca V 1.2/Ca V α2δ1/Ca V β3 subunits alone gave a current that exhibited a channel Po of 0.94 ± 0.01 (n = 6), while co-expression with 4R0N-tau produced a Ca V 1.2/Ca V α2δ1/Ca V β3-mediated current with a channel Po of 0.93 ± 0.03 (n = 7) (p = 0.91) (Fig. 5G).
Tau isoform-specific augmentation of L-type channel current. Human tau has 6 isoforms, 3 containing 3R repeats, while the remainder contain 4R C-terminal repeats (3R and 4R). Each of the two groups contain isoforms that possess either no, one or two near N-terminal inserts (0N, 1N and 2N). It has become apparent that 4R isoforms of tau are implicated in dementia 24   www.nature.com/scientificreports/ L-type channel current. Co-expression of 4R2N-tau produced a trend to augment L-type currents in tSA-201 cells transfected with Ca V 1.2, Ca V α2δ1 and Ca V β3 subunits (Fig. 6A-C). Peak current amplitude increased from − 92.5 ± 7.7 pA/pF in control (n = 9) to − 142.9 ± 28.5 pA/pF when co-expressed with 4R2N-tau (n = 10) (p = 0.06) ( Fig. 6A-C). Current was evoked using Ba 2+ as the charge carrier, showing that any effect of co-expression of tau is not dependent on the identity of the charge carrier. As was observed with the 4R0N-tau isoform, augmentation of macroscopic current by co-expression of 4R2N-tau was not accompanied by a shift in the voltage dependence of activation (control V 0.5 − 4.8 ± 2.7 mV, 4R2N-tau-expressing V 0.5 − 5.4 ± 4.0 mV) (Fig. 6D). A trend of an increase in the amplitude of current mediated by Ca V 1.3, Ca V α2δ1 and Ca V β3 subunits by co-expression of 4R2N-tau was seen in a similar way as observed co-expression of 4R0N-tau (Fig. 6E-G), and not seen when the channel contained the Ca V β2a subunit (Fig. S4). Peak current amplitude was non-significantly increased from − 104.9 ± 15.5 pA/pF (n = 8) to − 128.6 ± 9.0 pA/pF (n = 8) (p = 0.2). Finally, co-expression of 4R1N-tau and either Ca V 1.2 or Ca V 1.3, with Ca V α2δ1 and Ca V β3 subunits, failed to have any effect on the amplitude of functional current (Fig. 7) 0.3)). In addition, co-expression of 4R1N-tau had no effect on the voltage sensitivity of current activation (Fig. 7D,F)  These data illustrate that augmentation of macroscopic L-type current is dependent on the 4R isoform of tau, with the greatest effect observed upon co-expression of 4R0N-tau. In addition, there is some selectivity of the effect of 4R isoforms of tau, with greater enhancement of current observed with channels containing the Ca V 1.2 subunit.
Tau isoform-specific association with Ca V β3 subunits. Tau binds to microtubules to promote assembly and stabilization 32 . Tau has been also shown to directly interact with other proteins. For example, 4R0N-tau binds to Synaptogyrin-3 and thereby associates with presynaptic vesicles 33 , while 4R2N-tau interacts directly with heat shock protein (HSP) 90 34 and 14-3-3 proteins 35 . We used a co-immunoprecipitation approach to determine whether the tau-isoform dependent augmentation of functional L-type channel current results from a direct interaction. The augmentation of current by co-expressed 4R0N-tau is dependent on the channel containing the Ca V β3 subunit, therefore cells were transiently transfected with plasmids encoding either 4R0N-tau or 4R1N-tau, and the Ca V β3 subunit. Cell lysates were incubated with control IgG or tau antibody for co-immunoprecipitation, followed by Western blot analysis with anti-tau or anti-Ca V β3 (Fig. 8, see Fig. S5 for uncropped blots). Incubation with control IgG did not result in immunoprecipitation of any expressed protein, whereas anti-tau effectively immunoprecipitated 4R0N-and 4R1N-tau. Ca V β3 showed a robust and specific interaction with 4R0N-tau, in contrast to a significantly weaker interaction with 4R1N-tau (Fig. 8A). Data normalised for Ca V β3 expression and tau immunoprecipitation showed a 64% reduction in the level of Ca V β3-bound 4R1Ntau compared to 4R0N-tau (n = 3, t-test, p < 0.01) (Fig. 8B). This greatly reduced association between 4R1N-tau and the Ca V β3 subunit is consistent with the lack of a significant augmentation of functional Ca V 1.2, Ca V α2δ1, Ca V β3-mediated current (Fig. 7).

Discussion
The entry of Ca 2+ through L-type channels has been shown to activate the potassium channels that underlie generation of the Ca 2+ -dependent components of the medium and slow AHPs 11,27 . Expression of a 4R isoform of human tau augmented both AHP components, an effect resulting from an increase in the amount of Ca 2+ entry through L-type channels (Figs. 1, 2, 3). This increase in Ca 2+ entry (Fig. 4) resulted from an increase in the number of functional channels in the membrane (Fig. 5). The effect of expression of tau was not seen with 4R1N-tau, but was observed with both 4R0N and 4R2N isoforms (Figs. 4, 6, 7). L-type Ca 2+ channels are multisubunit complexes and require a beta subunit that regulates trafficking and biophysical properties of the functional channel. The effect of tau expression to augment macroscopic L-type channel current was only observed when channels possessed the Ca V β3 subunit (Fig. 4) and not when expressed channels contained the Ca V β2a subunit (Fig. 5). Finally, the identity of the α1 pore-forming subunit affected the magnitude of enhancement of macroscopic L-type current by expression of 4R0N and 4R2N isoforms of tau. L-type channels containing the Ca V α1.3 subunit were less sensitive to co-expressed 4R0N-or 4R2N-tau than channels containing the Ca V α1.2 subunit, despite both containing the Ca V β3 subunit (Figs. 4, 6).
Co-expression of channels and membrane associated proteins can affect macroscopic current. For example, sodium channel expression increases with co-expression of the microtubule-associated protein Map1b 36 . The auxiliary Ca V α2δ3 subunit associates with neurexin (NRX-1) to decrease Ca V 2.2-mediated whole-cell current 37 . Co-expression of Rab interacting molecules (RIMs) 2α and 3γ augmented Ca V 1.3/Ca V β2a/ Ca V α2δ1-mediated current by directly interacting with the C-terminus of the α1 pore-forming subunit 38 . Our data suggests that the physical association between 4R0N-tau and Ca V β3 results in a stabilization of functional Ca V 1.2-containing channels in the membrane, leading to augmented Ca 2+ entry. In addition, we observed a similar augmentation of evoked AHPs in hippocampal neurons expressing 4R0N(P301L)-tau (data not shown). This mutation within tau that is present in Hereditary Frontotemporal Dementia and Parkinson's disease which is linked to chromosome 17 (FTDP-17) augments co-expressed L-type channel current 39 . Mice over-expressing this mutation (P301L) exhibit a larger AHP in dorsal entorhinal cortical neurons 40 . Hippocampal neurons isolated from older animals displayed greater protection by nimodipine from Ca 2+ -mediated excitotoxicity than neurons from younger animals 41 , consistent with a reported increase in L-type channel current in hippocampal neurons from older animals 18  www.nature.com/scientificreports/ 1.3 subunits, which is countered by more channels in the membrane as measured by surface biotinylation 19 . It is proposed that this increase in L-type channel current underlies the loss of hippocampal neurons as a common neuropathological feature in old age 42 . The expression of tau increases in age 43 and we propose that the demonstrated physical interaction with tau stabilizes L-type channels in the membrane to augment Ca 2+ entry. We consider that this enhanced entry of Ca 2+ augments AHPs in hippocampal neurons, inhibiting action potential firing and possibly producing cognitive deficits 15 . The presence of amyloid β42 and tau proteins in cerebrospinal fluid (CSF) are core biomarkers for the diagnosis of AD 44 . The levels of tau are elevated in the CSF of sufferers of AD and there is evidence that a greater proportion of 4R-tau isoforms are present 45 . Normal human adult hippocampus contains approximately equal amounts of 3R-tau and 4R-tau isoforms 46 , with a shift in the ratio being proposed to result in early cognitive impairments 47,48 . For example, it has been suggested that an excess of 4R-tau relative to 3R-tau isoform produces neurodegeneration in Drosophila 49 . Abundance of 4R isoforms of soluble tau have been reported in patients with Alzheimer's disease, progressive supranuclear palsy and Pick's disease 50 . It is plausible that the increased number of L-type Ca 2+ channels in hippocampus of humans diagnosed with AD 51 results from association of the channel with elevated expression of 4R-tau isoforms.
Dysfunction of how the levels of Ca 2+ are regulated within a neuron has been implicated in AD and tauopathies 52 . For example, the intracellular Ca 2+ level within cortical neurons of a mouse transgenic model of AD (3xTg-AD) was twice that observed in control cortical neurons, and this dysfunction was caused by both changes in Ca 2+ entry and release from internal stores 53 . Our data suggest a mechanism for the regulation of cellular Ca 2+ levels by over-expression of a 4R-tau isoform. We propose that the demonstrated interaction between the 4R-tau isoform and Ca V β3 subunit results in an increase in the number of functional L-type Ca 2+ channels in the membrane.
It is certain that cognitive impairment occurs prior to diagnosis of tauopathies, but it is not known whether this results from cell loss in defined brain regions. The mild cognitive impairment observed in the early stages of tauopathies may result from the increased surface expression of functional L-type Ca 2+ channels. This increase has been shown to produce an enhanced AHP following a train of action potentials that would inhibit subsequent firing. This enhancement of the AHP mirrors that seen in aged animals, where suppression of the AHP by a L-type channel blocker aids learning 14 . It is suggested that a similar enhancement produced by wild-type tau expression would produce mild cognitive impairment. Finally, AD and tauopathies are characterized by cell loss, a process that has been proposed to involve dysregulation of intracellular Ca 2+ levels. The increased level of intracellular Ca 2+ within cortical neurons of a mouse transgenic model of AD (3xTg-AD) was largely reduced by www.nature.com/scientificreports/ inhibition of L-type Ca 2+ channels 53 . These data suggest that stabilization of L-type Ca 2+ channels in the plasma membrane by association with tau could result in both early cognitive impairment and contribute to cell loss in the later phase of AD or tauopathies.

Methods
Organotypic hippocampal slice cultures. Organotypic  www.nature.com/scientificreports/ Data analysis. AHPs were elicited by evoking a train (15) of APs by brief (2 ms) 2 nA somatic current injections delivered at 50 Hz. Any cell that did not fire the correct number of APs was discarded. Analysis of the medium and slow AHPs was carried out using custom-written MatLab scripts (The MathWorks Company). The medium AHP was measured as the peak negative membrane deflection between 0 to 100 ms after the cessation of the last AP fired. The slow AHP was measured 1 s after the last AP was fired. The overlapping kinetic profiles of the medium and slow AHPs is minimized by measuring the medium AHP and slow AHP at these time points 27 . All recordings used cells with a stable resting membrane potential more negative than − 60 mV. For voltage clamp recordings, data was analysed using Pulsefit (HEKA, Lambrecht/Pfalz, Germany). I mAHP was measured from zero to peak current, while I sAHP was measured 1 s after the end of the depolarizing voltage step.
Drugs. All drugs were bath applied. NBQX was prepared as a stock solution in dimethylsulfoxide (DMSO) and diluted in aCSF when required. All salts were purchased from Sigma-Aldrich except HEPES, which was obtained from Merck Serono (Fletham, UK).
TsA-201 cell electrophysiology. The tsA201 cell line was maintained as described previously 27 . Cells were transiently transfected using Lipofectamine LTX with plasmids encoding Ca V 1.2 (GenBank™ accession number AF394940) or Ca V 1.3 (GenBank™ accession number D38101), with either Ca V β2a (GenBank™ accession number M80545) or Ca V β3 (GenBank™ accession number M88751) and Ca V α2δ1 (GenBank™ accession number AF286488), with eGFP expression used as a marker for transfection. Using fluorescent proteins as a marker for ion channel expression is used widely and enables targeted recording without affecting channel function 55,56 . Cells were transfected while seeded in 25 cm 2 flasks, using 1.2-2 μg of each channel subunit plasmid and 0.4 μg of plasmid encoding eGFP in 8 mls of DMEM. Experiments to resolve the effects of co-expression of a 4R-tau isoform were accomplished by using 2 μg of construct in addition to those listed above. Cells were seeded into 35 mm culture dishes 24 h after transfection and cells expressing enhanced green fluorescent protein were used for electrophysiology 24 h later.
Expressing cells were bathed in an external solution of composition (mM): TEACl, 140; HEPES(Na), 10; CaCl 2 , 10; MgCl 2 , 1 and D-glucose, 10 (pH 7.4, 320 mOsm) and whole-cell voltage-clamped using electrodes manufactured from KG-33 borosilicate glass containing (mM): CsCl, 120; TEACl, 20; HEPES(Na), 10; EGTA, 5; Na 2 ATP, 1.5 and MgCl 2 , 1.5 (pH 7.4, 280 mOsm). Electrode resistances were 2-5 MΩ. Membrane current was recorded using an Axopatch 200A and filtered at a cut-off frequency of 1 kHz and sampled at 10 kHz using Pulse (HEKA). Currents were evoked by step depolarizations from a holding potential of − 80 mV, with 70-90% series resistance compensation used throughout. Evoked currents were leak subtracted using a P/4 protocol from a holding potential of − 90 mV. Non-stationary noise experiments were conducted using Ba 2+ as the charge carrier, where the external solution had an identical composition but where 10 mM BaCl 2 was used in place of CaCl 2 . A single pulse protocol of a 200 ms step depolarization to + 10 mV (peak of the current-voltage relationship) imposed every 5 s and repeated for a minimum of 100 sweeps.
Co-immunoprecipitation. TsA201 cells were grown in 6 cm dishes until 80% confluency was achieved. www.nature.com/scientificreports/ for significance were conducted using a non-paired Students t-test. Mean current values were calculated from individual cells and presented as ± standard error of the mean (SEM).

Data availability
All materials, data and associated protocols will be made available to readers upon reasonable request to Prof. N.V. Marrion (N.V.Marrion@bristol.ac.uk), without undue qualifications.